CA2066728C - Anti-tumor preparation comprising interleukin-2 and histamine, analogs thereof or h2-receptor agonists - Google Patents

Abstract

A preparation or system for inhibiting the development of malignant tumors and the formation of metastases of malignant tumor cells comprises a first composition comprising an agent selected from the group consisting of histamine, analogs thereof having H2-receptor activities, endogenous histamine releasing preparations and H2-receptor agonists, and a second composition comprising IL-2, said first and second compositions being either mixed in a preparation or provided in separate doses in an amount sufficient for treatment of tumors and metastases of malignant tumor cells.

Description

N
WO 91/04037 PC.T/SE90/00599 ANTI-TUMOR PREPARATION COMPRISING INTERhEUKIN-2 AND
HISTAMINE, ANALOGS THEREO>; OR H,-RECEPTOR AGONISTS
S ~'echnical Field This invention relates to the field of anti-tumor therapy, and more particularly to the treatment of malignant tumors with itaterleukin-2 (IL-2). The improvement provided by the present .invention is the coadministration of the IL-2. with an agent such as histamine, analogs thereof having HZ-recep-tor activities, endogenous histamine releasing preparations or HZ-receptor agonists. Unexpectedly potentiated effects are observed in the killing of tumor cells by components of immune system and the prevention or inhibition of metastases of tumor cells.
Background ~5rt Histamine has been shown to suppress a variety of immune effector mechanism.in vitro. This property of histamine is HZ-receptor associated. This effect' has been described in the literature as being either directly or indirectly mediated.
The direct effect is exerted via the CAMP-mediated suppres-sion of immunocompetent cells. The indirect effect is me-diated via the formation of histamine-induced suppressive proteins by suppressor T cells (see, Heer, D.J. et. al, Adv.
Immunol. 35: 209 (1984)).
The cancept that histamine may provide a suppressive signal for immune effector cells has also provided the background for other types of studies. One example is the testing of the potential antineoplastic effect of cimetidine and other HZ-receptor blockers, alone or in combination with other antineoplastic agents. Results of tests on the effects of these agents on tumor formation which have been conducted in rodents and humans are, however, conflicting. On one hand, ~~~~ i'~~
~'O 9i/0403i PC.'T/S E90/00599 the administration of HZ blockers has been reported to suppress tumor development in rodents and human subjects (see, e.g., Osband, M.E. et.al, Lancet 1(8221): 636 (1981). , Other studies, on the other hand, report that the same treatment enhances tumor growth and even induces tumors (see, e.g., Barna, B.P. et. al, Oncology 40: 43 (1983)).
Histamine has also been shown to suppress rather than enhance the growth and occurrence of several types of tumors (see, e.g., Burtin, C. et. al, Cancer Lett. 12: 195 (1981)).
The mechanism for the anti-tumor effects of histamine is not known but has been attributed to H1 receptor activity (see, e.g., Lespinats, G. et. al Br. J. Cancer 50: 545 (1984)).
Again, contradictory data exist in th9.s area as well. Hista-mine, for instance, has been reported to accelerate tumor growth in rodents (Nordlund J.J. et. al J. Invest. Dermatol 81: 28 (1983))~
Interleukin-2 (IL-2) is a lymphokine which has been ascribed a pivotal role in the expansion of T cells in response to antigen (Smith, K.A.~Science 240: 1169 (1988)). IL-2 has bean shown to exert anti-tumor effe~~ts in rodents (sae e.g., Lotze, M.T. et. al, in "Interleuki;n 2", ed. K.A. Smith, Academic Press Inc.,',San Diego, CA p. 237 (1988), Rosenberg, S., Ann. Surgery 208: 121 (1988)). IL-2 has also been shown to induce partial regression of established tumors in pati-ents with different types of cancer ~(Rosenberg, S.A. Ann.
Surgery 208: 121 (1988)). The anti-tumor effect of IL-2 is potentiated when the compound is given together with auto-logous lymphocytes which have been cultured in vitro with IL-2 and subsequentially been reinfused to the patient (lymphokine-activated killer (LAK) cells) (Rosenberg, S.A., Ann. Surgery 208: 121 (1988)). This effect is seen both in rodents and in humans. When used in human anti-cancer tri- ' als, IL-2 is usually given at very high doses to human tumorbearing subjects and has been reported to induce se-rious side effects, including renal disturbances, anemia, reduced platelet counts, and cardiorespiratory effects. In several of these trials the Hz-receptor antagonist ranitidine was used to prevent TL-2 induced dyspepsia and nausea (Rosenberg, supra).
NK cells are cansidered to play an important role in a host's defenses against arising neoplasms as well as against metastases (Hanna, N., Sur. Synt. Pathol. Res. 2: 68 (1983);
Hanna, N. Biochim. Biophys. Acta 780: 213 (1985)). Activa--tion of NK cells, in turn, is known to increase a host' s resistance against tumor cells (see, e.g., Lotze, M.T.
et.al., supra).
The following are individual in vitro effects of histamine and IL-2 on,the regulation of human NK cells known at the time of this invention.
(1) Histamine augments human NK cell cytotoxicity (NKCC) via HZ-receptors .
Histamine, at concentrations of 10-'-10-6 M, has been shown to strongly augment the NKCC of human mononuclear cells (MNC) against K562 leukemic cells. The effects is noted both when the ef~ector cells used are unfractionated MNCs or cells enriched for large granular lymphocytes (LGL) by Percoll density gradient centrifugation. The NK-augmenting response to histamine is also mimicked by the. Hz-receptor agonist dimaprit with similar potency and efficacy. Two structural analogues to dimaprit, nor-dimaprit and N-methyl--dimaprit, both lacking activities a-t Hz-receptors , proved to be in-effective under the same test conditions. The NK-augmenting effects of histamine and dimaprit were to be completely antagonized by the HZ-receptor antagonists ranitidine and cimetidine. The NK-augmenting effect of histamine was shown to require the presence of monocytes. In the absence of monocytes, histamine had no effect or weakly suppressed NKCC
at the histamine concentrations mentioned. (Hellstrand. K., ~~~~~1~~3 et.al, J. Immunol. 137: 656 (1986)).
(2) Histamine suppresses NK cell activity via T cells.
In contrast to the above-mentioned NK cell activation in-duced by histamine in the presence of monocytes, histamine has been reported to suppress NKCC against K 562 cells in the presence of T lymphocytes. Thus, in vitro treatment of human T cells with histamine (10'3 - 10-eM) induces the production of a soluble factor, histamine-induced soluble suppressar factor (HISSF) that inhibited NK cell cytotoxi-city. NK cells alone do not produce HISSF. Praduction of HISSF induced by histamine is blocked by cimetidine but not by an H1-receptor antagonist. The inhibition of NK cell cytotoxicity by HISSF is reduced by the addition of IL-2 (6.4-64 U/ml) or interferon-a (500 U/ml) (Hair, M.P.N.
et.al., J. Immunol. 136:2456 (1986)). Further, it has been shown that the T-cell mediated suppressive effect of hista-mine on NK-cell related cytotoxicity is more pronounced in.
the presence of IL-2 (Welt, S. et.al., Proc. Annu. Meet. Am.
Soc. Clin. Oncol: 7:A632 (1988)).
(3) Enchancement of NK cell cytotoxicity by IL-2.
IL-2 rapidly and effectively augments the cytotoxicity of isolated human NK cells in vitro over a broad range of concentrations. The effect has been: described both with natural and recombinant forms of IL-2 (Dempsey, R.A., et.-al., J. Immunol. 129:2504(1982); Phillips, J.H., et.al., J.
Exp. Med. 170:291 (1989)). The NK-augmenting effect of IL-2 is related to a cellular IL-2 receptor (IL-2R), p 75 (IL--2Ra) which is expressed on human NK cells (Siegel, J.P.
Science 238:75 (1987); Phillips, J.H., et.al., supra). The effect of TL-2 on NK cells is of relevance for the anti-tumor effect induced by this compound since depletion of NK
cells from mice was reported to eliminate anti-tumor effects induced by IL-2 treatment (Lotze, M.T., et.al., supra).

In view of the high incidence of cancer in the human population and the at best partial success obtained at present with the different therapies in existence, there is still a need for further improved methods of treating tumors in humans.
Disclosure of the Invention This invention relates to a preparation or system for inhibiting tumor growth and the formation of metastases of malignant tumor cells comprising a first composition comprising IL-2 and a second composition comprising an agent selected from the group consisting of histamine, analogues thereof having HZ-receptor activities, endogenous histamine releasing preparations and HZ-receptors agonists, said first and second compositions being either mixed in a preparation or provided in separate, doses in an amount sufficient for treatment of tumors and metastases of malignarnt tumors.
According to an object of an aspect of the present invention there is provided a pharmaceutical preparation or system for inhibiting tumor growth and the metastases of malignant tumor cells, characterized in, a first agent selected from the group consisting of histamine, analogues thereof having H2-receptor activities, endogenous histamine releasing preparations and HZ-receptor agonista and a second agent comprising IL-2, said first and second agents being either mixed in a preparation or provided in separate doses in an amount sufficient for treatment of tumors and metastases of malignant tumor cells.
According to another objecl: of an aspect of the present invention there is provided use of a first composition comprising a first agent selected from the group consisting of histamine, analogues thereof having Hz-receptor activities, endogenous histamine releasing preparations and HZ-receptor agonists and a second agent comprising IL-2 for preparing a pharmaceutical preparation or system for inhibiting tumor growth and the metastases of malignant tumor cells, said first and second agents being either mixed in a preparation or provided in separate doses in an amount sufficient for treatment of tumors and metastases of malignant tumor cells.
A more complete appreciation of the; invention and many of the attendant advantages thereof will be readily perceived as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying figure.

5a Brief Descr~tion of the Drawing The sole figure is a histogram showing the number of lung metastatic foci of B
16 melanoma cells produced by various treatments of male mice. The treatments were conducted with a vehicle (c, control), 25 mg/I:g histamine (h), 6x103 U/kg human recombinant IL-2 (IL), 25 mg/kg + 6x103 U/kg human recombinant IL-2 (h+IL), 25 mg/kg ranitidine (r), 6x103 U/kg human recombinant IL-2 + 25 mg/k~; ranitidine (r+IL). The compositions were injected to a 4-6 week old male Swiss albino mice and l.Sx105B16 melanoma cells were injected i.v.

~~~'~?~3 WO 91104037 ~CT/SE90/00599 to the mice 24 hours later. Treatment with vehicle, hista-mine, IL-2, ranitidine, histamine + IL-2, and rantidine _ IL-2 was repeated 1 week after tumor inoculation. The lung , mestatic foci (LMF) ware monitored after sacrifice of the animals 21 days later. Open bars represents the mean number of LMF on the lung surface calculated from 10 animals per treatment. Similar results were obtained in two separate experiments. The filled bars show lung weights of the respective' treatment groups. The weights of lungs correlated to the number of LMF. A seen in the figure, the lung weight of animals treated with histamine + IL-2 was equal to that of normal, tumarfree lungs.
Other objects, advantages and features of the present in-vention will become apparent to those skilled in the art from the following discussion.
Best Mode for Carrying out the Invention This invention arose from the rinexpected in vitro findings that (i) IL-2 can suppress NKCC in the presence of monocytes, and (ii) histamine and IL-2 act synergistically with respect to NKCC enchancement.
These findings prompted the inventors 'to analyze the in vivo effects of combined histamine/IL-2 treatment on the for-mation of lung metastases in a mouse animal model.
Unexpectedly, a combined histamine/IL-2~treatment completely prevented metastases of malignant tumor cells when the compounds were given as a single dose 2.4 hrs prior to and one week after tumor cells inoculation. These are unexpec- ' telly superior results since under similar circumstances neither IL-2 alone nor histamine alone had such beneficial effect. The doses of IL-2 used in the animal experiments WO 91!04037 ~ ~ PCT/SE90/005)9 were substantially lower than amounts used in general for treatment of cancer. This is of particular a.mportance since the potentiation of the anti--'tumor effect of IL-2 induced by concomitant treatment with histamine permits a reduction of the high doses of IL-2 which are used in cancer therapy.
Such high-dose. IL-2 treatment is associated with serious side-effects (Rosenberg, S.A., supra).
Provided herein is a preparation or system for inhibiting tumor growth and the metastases of malignant tumor cells in a subject carrying the cells comprising a first composition comprising an agent selected from the group consisting of histamine, analogues thereof having F32-receptor activities, endogenous histamine releasing preparations and HZ-receptor agonists and a second composition comprising IL-2; said agent and said IL-2 being either mixed~in a preparation or provided in separate doses in an amount sufficient for treatment of tumors and metastases of malignant tumor cells.
Analogs of histamine having H2-receptor activities which are suitable for.use in this invention are known in the art and need~not be described herein. By means of example, the analogs may hav a chemical structure similar t'o that of histamine~but be modified by the addition of moieties which do not negatively interfere with their histamine-like ac-tivities, and in particular with their Hz-receptor activiti-es. Examples of HZ-receptor agonists suitable for use in this invention are those such as dimaprit but not N-methyl-dima-prit or nor-dimaprit. Endogenous histamine releasing prepa-rations suitable for use herein are known in the art. Exam-ples of preparations capable of releasing endogenous hista-mine are these comprising other lymphokines such as IL-3 or allegens. However, other knwon preparations are also suit-able.
IL-2 and compounds such as histamine, analogs 'thereof, endogenous histamine releasing preparations and H~-receptor WO 91/fl4fl3i ~ ~ PCT/SE9fl/flfl599 agonists can be administered separately or in the same composition. The administration can be attained by routes which are krxown in the art for these compounds and prepara-tions. By means of example they can be administered by local or systemic injection, or infusion, as is known in the art.
However, other means of adminstration are also suitable.
The present compounds may also be adminstered by the intra-peritoneal and other parenteral routes. Solutions of the active compound as a free acid or a pharmaceutically--accept-able salt may be administered in water with ox without a surfactant sash as hydroxypropyl cellulose. Dispersion are also contemplated such as those utilizing glycerol, liquid polyethylene glycols and mixtures thereof and oils. Anti-microbial compounds rnay also be added to the preparations.
Injectable preparations may include sterile aqueous solu-tions or dispersions and powders which may be diluted or suspended in a sterile environment prior to use. Carriers such as solvents or dispersion media containing, e.g., 2J water, ethanol polyols, vegetable ails and the like, i~ay also be added. Coatings such as lecithin and surfactants may be utilized to maintain the proper fluidity of the composi-tion. Isotonic agents such as sugars or sodium chloride may also be added as well. as products intended for the delay of absorption of the active compunds such as aluminium monoste-arate and gelatin. Sterile injectable solutions are prepared as is known in the art and filtered prior to storage and/or administration. Sterile powders may be vacuum dried or freeze dried from a solution or suspension containing them.
Any material added to the pharmaceutical composition should be pharmaceutically-acceptable and substantially non-toxic in the amounts employed. Sustained-release preparations and formulations are also within the confines of this invention.
Pharmaceutically-acceptable carriers as utilized in the context of this patent include any and all solvents, dis-W(a 91/()4037 ~ ~ ~ ~ ~'~ ~ ~ fCT/8E90/00599 persion media, coatings, antimicrobial agents, isotonic and absorption delaying agents and the like as is known in the art. All preparations are prepared in dosage unit forms for uniform dosage and ease of administration. Each dosage unit form contains a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect in asso-ciation with a required amount of pharmaceutical carrier.
Typically, the agent which encompasses histamine, analogs thereof, endogenous histamine releasing preparations and HZ-receptor agonis-ts may be administered in an amount of about 0.1 to 10 mg/day, preferably about 0.5 to 8 mg/day, and more preferably about l to 5 mg/day. However, other amounts may also be administered with I1-2 as can be tailored by a practitioner.
Although in the examples the compounds are administered as a sole dose it is understood that for anti-tumor therapies the compounds may be administered for prolonged,periods of time. Typically,., the treatment may be administered for periods of up to about 1 week, and even for periods greater than 1 month. In some instance after a period of anti-tumor treatment, the treatment may be discontinued. and then resu-med once again.
The IL-2 may be administered in an amount of about 1.000 to 300.000 U/kg/day, more preferably about 3,000 to 100.000 U/kg/day, and more preferably about 5.000 to 20.000 U/--kg/day, or otherwise as known in the art.
A daily dose may be administered as one dose or it may be otherwise divided into several doses if negative effects are observed.
In one preferred embodiment of the method, the histamine, analogs thereof having Hz-receptor activities, endogenous histamine releasing preparations or H2-receptor agonists and !~O 91/t)4t)37 ~ ~~ ~ ~,~ ~ P~ PCT/SE9t)/Ot)599 the IL-2 are administered on the same days, A still more preferred embodiment of the method of the invention is tine wherein the agent is histamine and 'the histamine is adminis-tered in trxe same composition with IL-2.

In another aspect of the invention it is provided herein a method of increasing the anti-tumor cell effect of IL-2 in a subject comprising co-administering to the subject a first composition comprising IL-2 and a second composition compri-10 sing an agent selected from the group consisting of hista mine, analogues thereof having the Hz-receptor activities, endogenous histamine releasing preparatios and HZ-receptor agonists; the agent and the IL-2 being administered in amounts and for a period of time effective to attain the desired effect.
As in the case of the prior method the agent arid the IL-2 may be administered separately or as a single composition.
Typically, the agent is administered in an amount of about 0.1 to.l0 mg/day, more preferably about 0.5 to 8 mg/day, and more preferablyabout 1 to 5 mg/day for a period of time of about 1 week to 1 month, and in some instances for a period greater than 2 months. The Il-2 ma:y be administered in an amount of about 1.000 to about 300:000 U/kg/day, more prefe-rably about 3.000 to 100.000 U/kg/day, and more preferably about 5.000 to 20.000 U/kg/day, for a period of~about 1 week to 1 month, and in some cases the treatment may be prolonged for a period greater than about 2 months. The treatment with the two compounds may be, discontinued for a period of time and then resumed as was described above. Other regimes and amounts may also be utilized.
Also provided herein is an improvement on a knwon method of treating a subject carrying a malignant tumor with a compo-s-ition comprising IL-2, the improvement comprising co-ad-ministering to the subject a composition comprising an agent selected from the group consisting of histamine, analogues ~'O 91/04037 ~ ~ c~ ~ P~CTYSE90/00599 thereof having HZ-receptor activities, endogenous histamine releasing preparations and Hz-receptor agonistsl the IL-2 and the agent being administered in amounts and for a period of time effective to potentiate the anti-metastatic effect of IL-2.
The agent may be administered in amounts as described above, or as an artisan with skill in the art can determine. Simi-larly, the IL-2 may be administered in amounts known in the art (higher than prescribed herein), as described herein ar as an artisan may determine to be suitable for specific applications. Typically, the agent may be administered for 5a period of time of about 1 week and in some cases for even longer periods of time. Similarly the IL-2 may be adminis tered for a period of time as is known in the art for speci fic types of turnors or about 1 week to 2 months, and in many instances for longer periods of time as well.
In a garticularly preferred embodiment of the method the agent and the IL-2 are administered on 'the same days for increased potentiation of theix mutual effects.
Also provided herein is an improvement on a method of in-hibiting tumor grbwth and the metastases of malignant tumor cells in a subject carrying the cells with a composition comprising IL-2, the improvement comprising co-administerixlg to the subject a composition comprising an agent selected from the group consisting of histamine, analogues thereof having HZ-receptor activities, endogenous histamine releasing preparations and HZ-receptor agonists, the agent being administered in amounts and for a period of tune effective to increase the anti-tumor effect of IL-2 and to prevent the metastases of the celJ_s.
Typically, the agent is administered in an amount of 0.1 to 10 mg/day, more preferably about 0.5 to 8 mg/day and more preferably about 1 to 5 mg/day. The IL-2 is administered as WO 91/04037 fC?/SE90/OOS99 known in the art or in an amount of about 1.000 to 300.000 U/kg/day, more preferably about 3.000 to 100.000 U/kg/day and more preferably about 5.000 to 20.000 U/kg/day. The two compounds may be administered separately or in the same camgosition as described above.
In one preferred embodiment the agent and the IL-2 are administered on the same days and as a sole composition.
This therapy may be continued for a period of up to about 1 week, and even for periods longer than about 9 weeks. Rest periods flanked by treatment periods may also be utilized.
The present methods may be utilized alone or in conjunction with other anti-cancer therapies as seen suitable by a practitioner.
Having now generally described this invention, the same will be better understood by reference to certain specific examp-les, which. are included herein for purposes of'illustration only and are not intended to be'limiting of the invention or any embodiment thereof,.unless so specified.
Examples Example 1: In vitro Studies with IL--2 and Histamine.
This example provides a study on the effects of histamine, ranitidine and recombinant IL-2 (25U/ml), alone or in com-bination, in the NK-cell,~CytO'toxiCity (NKCG) of human mono-nuclear cells (MNC).
The MNC were obtained from pheripheral venous blood of healthy blood donors and recovered by Ficoll-Hypag centri-fugation followed by Percoll density gradient fractionation as previously described (Hellstrand, K. et. al. J. Immunol.
13%: 656 (1986)). A low density Percoll fraction 8 used in the experiments contained approximately 30% monocytes arid bV0 91J04~37 ~ ~ PCT/5~90/00599 was enriched for. large granular lymphocytes (LGL).
NKCC was measured in a 5'Cr--release microcytotoxicity assay using K 562 erythroleukemic, Daudi H-lymphoblastoid, Molt-4 T cells, and Chang liver cells as -target cells (all malig-nant cells).
NKCC was determined in sextublicate as specific 5'-Cr-release at a MNC:target cell ratio of 15:1 or 30:1. The assays were performed in Iscove's' medium containing antibiotics and l00 human AB+ serum. Histamine, IL-2, and ranitidine or various combinations thereof (see Table below) were added at the onset of a 4 hr 5'Cr-release assay. Control cells were given vehicle only.
The results obtained were as follows. Histamine ( 10'4 - 10-'M ) augmented NK cellcytotoxicity against all types of tumor cells in the presence of monocytes. This effect was entirely blocked by equimolar concentrations of ranitidine.
Ranitidine alone did not aff~ot NKCC. In .the absence of monocytes, i.:e., after removal of monocytes by h hr incuba-tion of the MNC on a Petri dish or by carbonyl iron treat ment, histamiiae, ranitidine, or histamine plus ranitidine were devoid of effects at any concentration tested.
IL-2 (5-50 U/ml) alone was unexpectedly ineffective or even suppressed NKCC in the presence of monQcytes. After removal of monocytes, IL-2 strongly augmented NKCC dose-dependently over the same range of concentrations. Histamine, ranitidine or histamine plus ranitidine did not affect IL-2-induced enchangement of NKCC in monocyte-depleted MNC. However, histamine plus IL-2 yielded a strong synergistic enchance-ment of NKCC in the presence of monocytes against all tumor cell targets tested. This synergistic effect was entirely blocked by the presence of ranitidine. Results of a repre-sentative experi.rnent are shown i a Table below.

WO 91/04037 ~ ~ ~ ~ ~ ~ ~ fC1'/SE90/00599 Table: Demonstration of Synergistic Activation of , Human NKCC by Combined Treatment with Histamine and IL-2 NKCC (cell lysis ~ ) ø s.e.m.) against repective tumor target cells Treatment) K562 Daudi Chang Molt-4 medium 21.6_+1.2 3.9+1.1 17.4+1.1 11.8+0.5 Hist(10-~M) 35.9+0.9 12.8+1.0 32.1+2.0 43.2+1.5 IL-2(25U/ml) 12.0_+0.7 1.4+0.5 9.8+0.6 5.2+0.4 Ran (10'SM) 20.8+1.9 4.3+0.8 19.7+1.3 13.0-X1.0 Hist + IL-2 55.4+~..0 41.4+0.9 59.7+0.6 69.4+3.0 Hist + Ran 20.1+1.4 5.0+1.4 19.4+1.0 13.0+1.1 Ran + IL-2 11.3+1.3 1.9+0.3 10.4+0.9 6.4+0.7 His-t + Ran 13.4+2.0 2.0+0.7 10.0+0.5 8.0+1.1 + IL-2 1 Effector MNC were recovered from peripheral blood by Ficoll-Hypaque and Percoll density gradient centrifu gation. A l.ow density Percoll fraction with 27% mono cytes was used at a final effector to target cell ratio of 15:1~.(K562 Chang, and Molt-4) or 30:1 (Dau di).
2 Hist = histamine, IL-2 = interl~ukin--2 Ran - Ranitidine Example 2: In vivo Studies Model of Antitumor Effects of Histamine, IL-2, Rantidine and Combinations of these Compunds in a Mouse Tumor Animal Model.
In vivo experiments were carried out with histamine or IL-2 alone, and with combinations of these compunds in a mouse tumor animal model.

W~ 91/04037 ~ ~ ~ ~'~ ~ ~ fCl/51;90/(10599 Histamine (25 mg/kg), ranitidine (25 mg/kg), and human recombinant I1-2 (6.000 U/kg), alone ar in comlaination, were administered, as a single-dose to 4-6 weeks old male Swiss 5 albino mice ( 20 g.) 24 hours prior to and 1 week after intra-venous inoculation of B 1S mouse melanoma cells (150.OU0 cells/mouse). Each treatment group comprised 10 animals.
Twenty--four hours after treatment, NK-cell sensitive B16 mouse melanoma cells (150.000 cells/mouse) were inoculated 10 intravenously. Controls were run with animals treated with the respective drug vehicles.
Lung metas~atic foci (LMF) an the surface of the lungs were monitored macroscopically 21 days later. LMF were counted by 15 an unbiased observer using a 10 x magnifying microscope. All LMF visible an the lung surface were counted.
The weights of the lungs were measured. immediately after sacrifice of the mice and correlated in a virtually linear fashion to the number of LMF.
Example 3: Results of the Tests Conducted in Example 2.
Under the therapeutic regimen depicted in Example 2, hista--mine alone was found to relatively effectively reduce the number of LMF. 25 mg/kg of histamine yielded approximately an about 50% reduction whereas 250 mg/kg of histamine yiel-ded an about 80-90~ reduction of LMF.
This effect was mimicked by dimaprit with similar potency.
Ranitidine augmented LMF about 100p.
IL-2 alone reduces LMF by about 40-70%.
The combined treatment with histamine (25 mg/kg) and IL-2 completely prevented LMF (see the Figure). None of the animals (n=10) treated with histamine (25 mg/kg) + I1-2 (6x103 U/kg) displayed visible tumors. None of the animals ( n=10 ) treated with histamine ( 25 mg/kg ) or IL-2 ( 6x103 U/kg ) W~) 91/OA037 ~~ ~ ~) ~ ~ ''e ~ PC'f~/SE90/005)9 alone were completely free of visible tumors. IL-2, was virtually ineffective in the presence of rinitidine. The lung weights of animals receiving histamine plus IL-2 was equal to the weight of lungs from ,mice that had not been subject to tumor cell inoculatian. Histamine, IL-2 or hista-mine plus IL-2 was found nat to affect lung weight of ani-mals which did not receive tumor cells.

Claims (18)

What is claimed is:

1. A pharmaceutical preparation or system for inhibiting tumor growth and the metastases of malignant tumor cells, characterized in, a first agent selected from the group consisting of histamine, analogues thereof having H2-receptor activities, endogenous histamine releasing preparations and H2-receptor agonists and a second agent comprising IL-2, said first and second agents being either mixed in a preparation or provided in separate doses in an amount sufficient for treatment of tumors and metastases of malignant tumor cells.

2. A pharmaceutical preparation or system as claimed in claim 1, wherein the daily dose of said first agent is between 0.1 and 10 mg.

3. A pharmaceutical preparation or system as claimed in claim 1, wherein the daily dose of said first agent is between 0.5 and 8 mg.

4. A pharmaceutical preparation or system as claimed in claim 1, wherein the daily dose of said first agent is between 1 and 5 mg.

5. A pharmaceutical preparation or system as claimed in any one of claims 1 to 4, wherein the daily doses of IL-2 is between 1,000 and 300,000 U/kg.

6. A pharmaceutical preparation or system as claimed in any one of claims 1 to 4, wherein the daily dose of IL-2 is between 3,000 and 100,000 U/kg.

7. A pharmaceutical preparation or system as claimed in any one of claims 1 to 4, wherein the daily dose of IL-2 is between 5,000 and 20,000 U/kg.

8. A pharmaceutical preparation or system as claimed in any one of claims 1 to 7, wherein the first agent is histamine.

9. A pharmaceutical preparation or system as claimed in any one of claims 1 to 8, characterized in that it further comprises at least one pharmaceutically-acceptable carrier selected from the group consisting of solvents, dispersion media, coatings, antimicrobial agents, and isotonic and absorption delaying agents.

10. Use of a first composition comprising a first agent selected from the group consisting of histamine, analogues thereof having H2-receptor activities, endogenous histamine releasing preparations and H2-receptor agonists and a second agent comprising IL-2 for preparing a pharmaceutical preparation or system for inhibiting tumor growth and the metastases of malignant tumor cells, said first and second agents being either mixed in a preparation or provided in separate doses in an amount sufficient for treatment of tumors and metastases of malignant tumor cells.

11. Use as claimed in claim 10, wherein the daily dose of said first agent is between 0.1 and 10 mg.

12. Use as claimed in claim 10, wherein the daily dose of said first agent is between 0.5 and 8 mg.

13. Use as claimed in claim 10, wherein the daily dose of said first agent is between 1 and 5 mg.

14. Use as claimed in any one of claims 10 to 13, wherein the daily dose of IL-2 is between 1,000 and 300,000 U/kg.

15. Use as claimed in any one of claims 10 to 13, wherein the daily dose of IL-2 is between 3,000 and 100,000 U/kg.

16. Use as claimed in any one of claims 10 to 13, wherein the daily dose of IL-2 is between 5,000 and 20,000/kg.

17. Use as claimed in claim 10, wherein the agent is histamine.

18. Use as claimed in any one of claims 10 to 17, further comprising at least one pharmaceutically-acceptable carrier selected from the group consisting of solvents, dispersion media, coatings, antimicrobial agent, and isotonic and absorption delaying agents.